Methods: We analyzed the results from a prospective nationwide multicenter AF registry. Follow-up data were collected for 3 years. The main outcomes were SSE. Predictive models of the 3-year SSE of the COOL-AF model, the CHA2DS2VASc score, the CARS for the no-OAC group, and the mCARS for the OAC group were developed and evaluated by C-statistics, and calibration plots were created for the whole group, as well as for oral anticoagulant (OAC) users and no-OAC patients. Results: We studied 3405 patients . The incidence rates of SSE were 1.51 (1.26–1.78), 1.93 (1.39–2.60), and 1.37 (1.10–1.68) for all patients, no-OAC patients, and OAC patients, respectively. For the whole population, the COOL-AF score had a C-statistic of 0.697 (0.682–0.713), which was superior to the CHA2DS2-VASc [0.655 (0.639–0.671)]. For the no-OAC group, the CARS predicted SSE with a C-statistic of 0.685 (0.652–0.716), which was similar to the CHA2DS2-VASc [0.684 (0.651–0.7150] and COOL-AF models [0.692 (0.659–0.723)]. For the OAC group, the mCARS had a C-statistic of 0.687 (0.669–0.705) that was similar to the COOL-AF [0.704 (0.686–0.721)] and better than the CHA2DS2-VASc score [0.655 (0.637–0.674)]. Conclusions: The calculation of the individual absolute risks using the CARS and mCARS models can predict SSE in an Asian population. Small differences were evident between the COOL-AF and CHA2DS2-VASc scores.Background: The aims of this study were (1) to validate the CARS and mCARS methods in an Asian population with atrial fibrillation (AF) and (2) to compare the CARS and mCARS models for absolute risk using the COOL-AF method and CHA For the no-OAC group, the CARS model can be used to predict SSE with a C-statistic of 0.685 (0.652–0.716), similar to CHA2DS2-VASc score [0.684 (0.651–0.7150] and COOL-AF model [0.692 (0.659–0.723)]. For the OAC group, the mCARS had a C-statistic of 0.687 (0.669–0.705), which was similar to the COOL-AF [0.704 (0.686–0.721)] but better than the CHA2DS2-VASc score [0.655 (0.637–0.674)] for the prediction of SSE.2DS2-VASc, COOL-AF, and CARS models in the OAC-treated groups, as opposed to the observed risks in the no-OAC groups  risk on the Y-axis by dividing the predicted probability of each model into 10 equal groups. There were overestimation trends of the CHAC groups .2DS2-VASc score.The results of this prospective nationwide multicenter study in Thailand demonstrated that the CARS in patients who were not using OACs and the mCARS for patients using OACs can be used to predict SSE in Asian populations. The predictive ability of these models was equivalent to the COOL-AF prediction model and at least as good as the CHA2DS2-VASc score was designed for simplicity and for population-level assessments . However, the number of patients not using OACs in our study was relatively small (n = 837 or 24.6%), which might explain the lower C-statistics of our study in comparison with the original CARS study from Denmark [2DS2-VASc of 1. The CARS underestimated the risk of SSE in this group [(0.63 (0.13–1.81)]. Therefore, caution should be exercised when applying the CARS model with Asian populations with CHA2DS2-VASc scores of 1.The CARS model was developed by the national Danish AF registry by selecting only patients who were not on OACs . The CAR Denmark . The res2DS2-VASc score [0.655 (0.637–0.674)] for the prediction of SSE in OAC-treated patients. Our results suggest that the mCARS model can probably be used in Asian AF populations. Among patients with CHA2DS2-VASc = 0 who received OACs, the actual SSE risk was 0.6 (0.01–3.28) per 100 person-years, whereas the incidence rate was 0.48 (0.01–2.64) according to the COOL-AF model and 0.15 (0.01–0.80) according to the mCARS. This result indicates that the mCARS method underestimated the risk of SSE in Asian populations with CHA2DS2-VASc = 0 who received OACs. Previous studies from Taiwan [2DS2-VASc = 0 are higher than indicated by data from non-Asian populations.The mCARS model was developed by Ding et al.  using thm Taiwan  and Hongm Taiwan  have demThe purpose of this study was to determine whether the CARS and mCARS models, which were developed from the results of non-Asian populations, can be applied to Asian populations. The results of our study indicated that the CARS and mCARS models can be used for Asian populations with similar predictive values to the COOL-AF model and the CHA2DS2-VASc model. While CHA2DS2-VASc is a scoring-based system for risk prediction, the COOL-AF, CARS, and mCARS models provide absolute risk predictions. Despite the fact that CHA2DS2-VASc scores may be easier to use, models with absolute risk prediction are probably more accurate and may be more precise for communication with patients.2DS2-VASc scores above 2 points, 1984 (71.2%) were on OACs alone, 287 (10.3%) were on OACs plus antiplatelet medications, 415 (14.9%) were on antiplatelet medications alone, and 101 (3.6%) were taking no antithrombotic drugs. Overall, 2271 (81.5%) patients were on OACs. The rate of OAC use among patients with CHA2DS2-VASc scores ≥ 2 in our study was greater than the reported rate of the Asian population in the GARFIELD registry and similar to the rate of non-Asian populations. Despite the fact that patients with CHA2DS2-VASc scores ≥ 2 should be on OACs, there are many factors that influence decisions concerning OAC treatment, including fear of bleeding or a history of bleeding, especially since the main OAC used in our study was warfarin.Among 2787 patients with CHALeft atrial appendage occluders (LAAOs) are an alternative option for the prevention of strokes in AF patients, especially for those who cannot be on OACs. Recent data has shown good long-term results with acceptable complication rates, even in elderly populations . The proThere are some limitations of this study. First, whether or not OACs were used of may have changed with time. Therefore, the choice to use or not use OACs at the baseline may not represent the OAC status for the whole period of the study. Second, warfarin was the major OAC used in our study. Therefore, the results may not be fully generalized to other populations which mainly used NOACs. Third, 56 patients (1.6%) were lost to follow-up or withdrew from the study. Fourth, the adjustment of covariates was performed by the Cox proportional-hazard model during the creation of the COOL-AF model. The factors that are not independent predictors for SSE were eliminated during the Cox proportional-hazard model. However, the medications that were in the model were OACs. We did not include treatment of hypertension or dyslipidemia in the models. We believed that patients who received these medications may be at a higher risk and may make it difficult to interpret the model.2DS2-VASc scores.The calculation of the individual absolute risks using the CARS and mCARS models, which are based on using a risk factor-based approach, can predict SSE in an Asian population. Small differences were evident compared to the COOL-AF and CHA


Nanocrystals (NCs) have the potential to enhance the oral bioavailability of Class IV drugs in the Biopharmaceutical Classification System (BCS) due to the absorption of the intact crystals. The performance is compromised by the dissolution of NCs. Drug NCs have recently been adopted as solid emulsifiers to prepare nanocrystal self-stabilized Pickering emulsions (NCSSPEs). They are advantageous in high drug loading and low side effects due to the specific drug loading mode and the absence of chemical surfactants. More importantly, NCSSPEs may further enhance the oral bioavailability of drug NCs by impeding their dissolution. This is especially true for BCS IV drugs. In this study, curcumin (CUR), a typical BCS IV drug, was adopted to prepare CUR-NCs stabilized Pickering emulsions using either indigestible  or digestible  oils, i.e., IPP-PEs and SO-PEs. The optimized formulations were spheric with CUR-NCs adsorbed on the water/oil interface. The CUR concentration in the formulation reached 20 mg/mL, which was far beyond the solubility of CUR in IPP (158.06 ± 3.44 μg/g) or SO (124.19 ± 2.40 μg/g). Moreover, the Pickering emulsions enhanced the oral bioavailability of CUR-NCs, being 172.85% for IPP-PEs and 152.07% for SO-PEs. The digestibility of the oil phase affected the amounts of CUR-NCs that remained intact in lipolysis and, thus, the oral bioavailability. In conclusion, converting NCs into Pickering emulsions provides a novel strategy to enhance the oral bioavailability of CUR and BCS IV drugs. About 40% of drugs on the market and 70% of new chemical entities under development are poorly water-soluble . Poor soLipid-based formulations have been a traditional solution to the oral delivery of BC IV drugs ,15,16. TPickering emulsions are emulsions with solid particles at the oil–water interface ,25. TheyAlbeit an LBF, NCSSPEs may undertake different mechanisms in enhancing the oral bioavailability. Cargos in NCSSPEs are mainly adsorbed on the oil surface as NCs instead of being dissolved in the oil. Since the amount of drug NCs tremendously exceeds the solubilizing limit of the oil phase, the secondary colloidal vehicles from lipolysis may only solubilize a small part of the co-formulated drugs despite their excellent solubilization capacity. Most of the drugs during the lipolysis remain in the nanocrystal form. The absorption of detached NCs from NCSSPEs may be the main reason for the enhanced oral bioavailability of BCS IV drugs instead of the traditional mechanism in LBFs. In addition, the formation of NCSSPEs may impede the dissolution of drug NCs since they are adsorbed on the surface of oil droplets. NCSSPEs are thus supposed to further enhance the oral bioavailability of their nanocrystal counterparts.To evaluate the potential of NCSSPEs in enhancing the oral bioavailability of BCS IV drugs, CUR, a typical class IV drug with various pharmacological activities ,32, was Curcumin (purity > 97%) was purchased from TCI Shanghai Chemical Industry Co., Ltd. , and the internal standard  was purchased from Dalian Meilunbio. Co., Ltd. . Hydroxypropyl methylcellulose E5 (HPMC E5) was a gift from Shanhe Medical Materials Co., Ltd. . IPP was obtained from Guangzhou Hefu Chemical Technology Co., Ltd. . SO was obtained from Shandong Ruisheng Pharmaceutical Excipients Co., Ltd. . Sesame oil was obtained from Sigma-Aldrich  Trading Co., Ltd. . Campul MCM and Captex 355 were gifts from IMCD (China) Co., Ltd. . Glyceryl monooleate was obtained from KarmaChem . Fasted-state simulated intestinal fluid powder was purchased from Biorelevant . Pancreatin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. , and the activity of amylase and trypsin was 100–350 U/mg, while the lipase activity was 10–75 U/mg. HPLC-grade methanol was purchased from Fisher Chemicals (UK). Other solvents and chemicals were of analytical grade.Male Sprague Dawley rats (200 ± 20 g) were purchased from JieSiJie Laboratory Animal Co., Ltd. . The rats were housed in the animal facility  with unlimited access to food and water and fasted for 12 h but free to water prior to the experiment. Our animal experiments were approved by the institutional ethics committee of Shanghai Skin Disease Hospital and were performed in compliance with institutional guidelines .w/v) HPMC E5 solution and subsequently processed with a high-pressure homogenizer  for 15 cycles at 20,000 psi to obtain nanosuspensions. The nanosuspensions were pre-frozen under −80 °C for 4 h and then lyophilized for 48 h to obtain solid CUR-NCs. The powders were stored in hermetic containers before use.CUR-NCs were prepared with minor modifications to the previous method . In brieThe size, polydispersity index (PDI), and zeta potential of CUR-NCs were measured by a Malvern Zetasizer Nano . CUR-NC suspensions were diluted 10-fold by deionized water before tests. The diluted CUR-NC suspensions were added into folded capillary cells with liquid level over electrodes for zeta potential tests. The morphology of CUR-NCs was observed by scanning electron microscopy (SEM) . Before observation at an accelerating voltage of 1.0 KV, CUR-NC powder was sputter-coated with gold.g, 0.2 g supernatant was withdrawn and diluted to 10 mL with ethanol. The absorbance of CUR was measured with a UV-Vis spectrophotometer  at 425 nm to determine the solubility of CUR. The solubility of CUR in FaSSIF was also determined.Five milligrams of CUR were dispersed in 2 g of liquid oils . The oil suspensions were placed in a water bath at 37 °C and shaken at 100 rpm for 24 h . After centrifugation for 10 min at 5000× Various oils were mixed with CUR-NC suspensions (10 mg/mL) at a ratio of 5:1 and dispersed by high shear at 13,000 rpm for 5 min in a high-speed homogenizer . The obtained emulsions were diluted 2-fold with deionized water and then dropped onto microscope slides and covered with coverslips, which were eventually observed with optical microscopy  to screen oils with CUR-NC adsorptive capacity on their droplet surface.IPP, an indigestible lipid, and SO, a digestible lipid, were selected as the oil phase to prepare Pickering emulsions. CUR-NCs were dispersed in deionized water as the water phase and then mixed with the oil phase (IPP or SO). The water–oil mixture was then processed with high shear at 13,000 rpm for 5 min in a high-speed homogenizer  to obtain CUR-NCSSPEs. Parameters including CUR concentrations  and the volume ratios of water: oil phase  were screened based on the size of the Pickering emulsions measured by Mastersizer 3000 .The morphologies of IPP-PEs and SO-PEs were observed by optical microscopy , confocal laser scanning microscopy (CLSM) , and cryo-scanning electron microscopy (Cryo-SEM).IPP-PEs and SO-PEs were diluted 2-fold with deionized water, which were then dripped onto microscope slides and covered with coverslips. Observed with the optical microscope, images were captured.Prior to CLSM imaging, Nile Red solution  was used to label the oil phase of IPP-PEs and SO-PEs. Twenty microliters of Nile Red solution were fully mixed with 0.5 mL Pickering emulsions. The stained Pickering emulsions were then diluted 2-fold with deionized water, which were dripped onto microscope slides and covered with coverslips. The slides were then observed under CLSM.The fluorescence of CUR and Nile Red was excited at wavelengths of 488 nm and 561 nm, respectively. The resolution of the scan was set to 1024 × 1024 pixels.For Cryo-SEM, the method was according to a previous study . SamplesMale Sprague Dawley rats (200–220 g) were fasted overnight before administration but were allowed free access to water. CUR-NCs (n = 6), IPP-PEs (n = 6), and SO-PEs (n = 4) were intragastrically administered to rats at a dose of 100 mg/kg, respectively. Blood samples were collected at predetermined time intervals  post-dose and placed into pre-heparinized microcentrifuge tubes immediately. Blood samples were centrifuged at 4000 rpm for 10 min at 4 °C. Plasma was kept frozen at −20 °C pending analysis.v/v) formic acid  was added into the plasma, which was vortex-mixed for 5 min. Following centrifugation at 6000 rpm for 10 min, the organic layer was collected and evaporated under nitrogen flow at 45 °C. Sixty microliters of the mobile phase were added to the residues and vortex-mixed for 30 s to dissolve the extracted CUR. Following centrifugation at 12,000 rpm and 4 °C for 20 min, the supernatant was obtained for LC–MS/MS analysis.The liquid–liquid extraction method was used to extract CUR from the plasma. Briefly, a 10 μL of emodin solution (2.442 ng/mL), the internal standard, was mixed with a 100 μL plasma sample. Then, a 1 mL mixture of ethyl acetate and methanol containing 0.1% (AUC0–t), were obtained using compartmental analysis by the DAS 2.0 software package . The values of peak blood concentration (Cmax) and peak time (Tmax) were obtained directly from raw data. One-way ANOVA was used to test the differences between groups, and p < 0.05 was considered to be a significant difference.The main pharmacokinetic parameters, such as the area under the concentration–time curve (v/v) was pumped at a flow rate of 0.2 mL/min. The detection was performed by a Thermoscientific triple quadrupole detector. The mass spectrometer was operated with an electrospray ionization (ESI) interface in negative ionization mode and multiple reaction monitoring modes. The selected reaction monitoring of CUR and emodin was m/z 366.95/216.97 and m/z 268.96/224.99, respectively. The peak area ratio of CUR to emodin (R) was used for quantification. The linearity was observed within the concentration (C) range from 0.5525 to 11.05 ng/mL with a typical calibration equation: R = 0.0998C − 0.0368, R2 = 0.9966 . CUR was separated by an Agilent Hypersil GOLD C18  column at 25 °C. The mobile phase composed of methanol: water . The FaSSIF stood for 2 h at room temperature before use. Twenty milliliters of FaSSIF were utilized to dissolve 0.2 g of pancreatin, which was then centrifuged at 1000× g for 10 min. Each phase was separated and dissolved with ethanol. The content of CUR was measured with the HPLC.Before lipolysis, two milliliters of pancreatin solution were mixed with 20 mL of FaSSIF. The pH was adjusted to 6.5 with 0.1 M NaOH, which was thermostatically kept at 37 °C in a water bath and stirred at 800 rpm. Then, 1 mL of CUR-NCs, IPP-PEs, and SO-PEs, equivalent to 20 mg/mL CUR, were added to initiate in vitro lipolysis. The pH of the system was maintained at 6.5 with a titration of 0.1 M NaOH. Upon predetermined time intervals , five hundred microliters of sample were withdrawn and separated into different phases by centrifuging  at 1000× The prepared CUR-NCs represented a uniform rod shape, with a mean size of 272.13 ± 2.65 nm, PDI of 0.080 ± 0.001 , and ZetThe solubility values of CUR in oils and Biorelavent fluids are listed in The volume ratios of water to oil phase were set as 1:2, 1:1, and 2:1 to investigate their effects on the size and size distribution of IPP-PEs. The results showed that the sizes of IPP-PEs tended to decrease with the increasing proportion of water. When the ratio was at 1:1 and 2:1, D, D10, D50, and D90 were comparable and lower than those of 1:2 with lower SD values A. ConsidThe size distribution of IPP-PEs and SO-PEs is shown in The optical micrographs of IPP-PEs and SO-PEs are displayed in To confirm that CUR-NCs adsorb on the water/oil interface, the oil phase of the Pickering emulsions was stained using Nile Red for imaging with CLSM . Nile ReIn addition, the microstructures of IPP-PEs and SO-PEs were observed by Cryo-SEM . Both IPTmax and moderate Cmax among all preparations. Since CUR belongs to BCS IV drugs, the absorption may be attributed to transepithelial transport of the intact NCs instead of diffusion of the dissolved molecules. However, dissolution led to the reduction of intact NCs. After a quick absorption and reaching a moderate peak concentration, the plasma concentration of CUR decreased, producing the lowest AUC0–8 among all preparations. IPP-PEs achieved a similar pharmacokinetic profile to CUR-NCs except for a longer Tmax of 0.75 ± 0.25 h and a higher Cmax of 26.78 ± 3.54 μg/L. The curves in the absorption phase from the two preparations are almost superposed. It is inferred that transepithelial transport of detached CUR-NCs may be the main reason for the absorption of IPP-PEs. Since IPP is indigestible, the structure of Pickering emulsions may hinder the dissolution of CUR-NCs due to the interaction with the oil. The continuous transport led to the higher Cmax and the lag of Tmax. Consequently, the oral bioavailability of IPP-PEs reached 172.85% relative to CUR-NCs. SO-PEs achieved a different pharmacokinetic profile, presenting triple peaks. The first peak at 10 min may be due to the fast absorption of unadsorbed CUR-NCs in the preparation. CUR-NCs may have stronger interaction with SO than IPP. However, SO is digestible. CUR-NCs may be gradually detached from SO-PEs with the lipolysis of SO. The subsequent absorption of detached NCs led to multiple peaks. Therefore, SO-Pes achieved the longest Tmax among the preparations. Although SO-PEs achieved a lower Cmax than CUR-NCs, the AUC and oral bioavailability were enhanced due to the extended absorption period. Nonetheless, IPP-PEs had a higher oral bioavailability than SO-PEs, being 172.85% versus 152.07%. Of note is that the oral bioavailability is relative to NCs. An even higher bioavailability can be anticipated in comparison with CUR coarse crystals. The pharmacokinetics study proved the efficiency of Pickering emulsions in promoting oral absorption of CUR-NCs, which may apply equally to other BCS IV drugs.The plasma concentration–time curves of IPP-PEs, SO-PEs, and CUR-NCs are presented in The pH-stat lipolysis model was adopted to estimate the intestinal digestion of Pickering emulsions. The morphology transformation of IPP-PEs and SO-PEs during the lipolysis was shown in Tmax. To sum up, the NCSSPEs delayed the dissolution of CUR-NCs and provided more opportunities for the intact NCs to be absorbed, standing for a novel strategy to enhance the oral bioavailability of CUR and BCS IV drugs.CUR-NCs could be prepared via a homogenization method. Based on the solubility and interaction, IPP and SO were selected to prepare CUR-NC-stabilized Pickering emulsions, respectively. IPP-PEs and SO-Es were spheric, while CUR-NCs were adsorbed on the water/oil interface. Therefore, CUR concentration in the formulation was far beyond CUR solubility in IPP and SO. The Pickering emulsions enhanced the oral bioavailability of CUR-NCs by 1.73- (IPP-PEs) and 1.52- (SO-PEs) fold, respectively. The difference may be due to the digestibility of the oil phase. The indigestible IPP maintained a higher percentage of intact CUR-NCs than the digestible SO in in vitro lipolysis, leading to a higher AUC and a shorter


In the general population, an increase in low-density lipoprotein cholesterol (LDL-C) predicts higher cardiovascular disease risk, and lowering LDL-C can prevent cardiovascular disease and reduces mortality risk. Interestingly, in cohort studies that include very old populations, no or inverse associations between LDL-C and mortality have been observed. This study aims to investigate whether the association between LDL-C and mortality in the very old is modified by a composite fitness score.A 2-stage meta-analysis of individual participant data from the 5 observational cohort studies. The composite fitness score was operationalized by performance on a combination of 4 markers: functional ability, cognitive function, grip strength, and morbidity. We pooled hazard ratios (HR) from Cox proportional-hazards models for 5-year mortality risk for a 1 mmol/L increase in LDL-C. Models were stratified by high/low composite fitness score.p < .01), most pronounced in participants with a low-composite fitness score , compared to those with a high composite fitness score , the test for subgroups differences was not significant.Composite fitness scores were calculated for 2 317 participants , of which 994 (42.9%) had a high composite fitness score, and 694 (30.0%) had a low-composite fitness score. There was an inverse association between LDL-C and 5-year mortality risk (HR 0.87 [95% CI: 0.80–0.94]; In this very old population, there was an inverse association between LDL-C and all-cause mortality, which was most pronounced in participants with a low-composite fitness scores. Atherosclerotic cardiovascular diseases (ACVD) are highly prevalent in advanced age and an important cause of morbidity and mortality . LowerinHowever, observational research on the relation between LDL-C and all-cause mortality has offered conflicting results. Most observational studies performed in older populations report no, or inverse associations . These aTherefore, this study aims to investigate whether the association between LDL-C and mortality in the older is modified by fitness, operationalized with a composite fitness score. In this meta-analysis, we take the opportunity of combining individual participant data from several studies of people of advanced age, across nations, and cultural groups to examine the stability of associations around the world.This 2-stage individual participants data (IPD) meta-analysis includes data from 5 cohorts of older adults from the 4 studies of the Towards Understanding Longitudinal International older People Studies (TULIPS) Consortium: (1) the Leiden 85-plus Study; (2) Te Puāwaitanga O Ngā Tapuwae Kia Ora Tonu: Life and Living in Advanced Age, a cohort study in New Zealand (LiLACS-NZ), providing a (2a) Māori (Indigenous people of NZ), and (2b) non-Māori cohort; (3) the Newcastle 85+ Study; and (4) the Tokyo Oldest Old Survey on Total Health (TOOTH).The selection criteria for this analysis were the availability of either fasting or nonfasting LDL-C measurements at baseline. We excluded participants who had had a baseline triglyceride measurement of ≥4.5 mmol/L (or ≥400 mg/dL), as high triglyceride levels may distort LDL-C levels. To avoid effect overestimation due to the association between terminal illness and progressively declining LDL-C, we also excluded participants who died within the first 90 days of follow-up. For all studies, ethical approval was obtained from their respective authorities. Informed consent was obtained from all participants for data collection and publication of the results. Details on measures and data collection have been previously reported .In this study, all participants were recruited from the city of Leiden, The Netherlands. Of the 705 residents who reached the age of 85 between September 1997 and September 1999, 599 were enrolled in the study of which 556 had LDL-C levels measured at baseline .n = 1 636), 421 Māori and 516 non-Māori participants were enrolled , in 2010. Of the total age-eligible . There were 849 participants enrolled in the study with complete health assessment and record review , at basIn the LilACS-NZ study and Newcastle 85+ Study total cholesterol, high-density lipoproteins (HDL-C), LDL-C, and triglycerides were measured in venous blood samples after an overnight fast ,16. In tThere were 4 individual markers of fitness that were available in all cohorts, and that could be included in this study to define subgroups. Details on the variables used in the analyses are shown in 1) Functional ability: In the Leiden 85-plus Study, functional ability was measured with the Groningen Activity Restriction Scale  . In the 2) Cognitive function: In all cohorts, cognitive function was assessed with the Mini-Mental State Examination (MMSE) questionnaire (maximum score of 30). Higher MMSE scores indicate better cognitive functioning . A score3) Grip strength: Handgrip strength was measured in kilograms by the use of handheld dynamometers. Measurements of grip strength in the low sex-specific tertile (T1) cohort or inability to perform the test were considered a marker of low levels of fitness and in the high sex-specific tertile (T3) of cohort a marker of the high levels of fitness.4) Morbidity: Three chronic diseases with relevance for survival risk and LDL-C, that were assessed comparable in the 4 studies, were included: diabetes mellitus (diabetes), malignancy , and history of ACVD . Information on these diseases was obtained from the interviews and/or general practitioner records, and from electrocardiograms. We considered having ≥2 of these three morbidities (high morbidity), a marker of a low levels of fitness, and the absence of all 3 morbidities (low morbidity), a marker of high levels of fitness.Cutoff values for the tertiles of functional ability, cognitive function, and grip strength, were chosen in a way that the number of participants that fell in the groups was closest to 33% of the cohort size.We combined the 4 individual markers of fitness  into the composite fitness score, and pragmatically defined a low-composite fitness score as having ≥2 of the 4 markers of low level of fitness, and a high composite fitness score as having ≥2 of markers as high level of fitness. Participants that did not meet these criteria, or who had 2 individual markers as high and 2 individual markers as low level of fitness, were classified as rest group. These criteria best served the prespecified aim to compare participants with 33% highest and 33% lowest level of fitness in each cohort.For the Leiden 85-plus Study, LilACS-NZ study and the Newcastle 85+ Study, mortality data were obtained from national registries ,15,17. FIn the first stage, the analyses were performed at the cohort level. In the second stage, we pooled the cohort-level results.SD) or as median with interquartile range (IQR) depending on the distribution of the data. Categorical variables are presented as frequencies with the percentage of the valid total. To test for group differences between categories of participants, we used independent t tests or Mann–Whitney U-tests for continuous variables, and the chi-square tests for categorical variables. We carried out a 5-year survival analysis using Cox proportional hazards regression models. We calculated hazard ratios (HRs) with 95% confidence intervals (CIs) per mmol/L change in LDL-C level, adjusted for age, sex, and use of lipid-lowering medication .We present continuous variables as mean with standard deviation  and finally by the composite fitness score (high/low). We tested for subgroup differences with the Chi-square test between the strata.2-statistic, I2 below 40% was defined as consistency between estimates  we assessed whether the association between LDL-C and mortality was similar for participants with and without ACVD, by stratification, (2) to have a closer look at the role of lipid-lower medication use in the association between LD-C and mortality in the subgroups of the composite fitness score, we double stratified by composite fitness score (high/low) and by lipid-lowering medication use (yes/no), and adjusted for age and sex, and (3) we repeated the initial analyzes with total cholesterol instead of LDL-C. These analyses were also performed in a 2-stage approach.SD 1.1), the mean HDL was 1.5 mmol/L (SD 0.4), and the mean LDL-C was 3.0 mmol/L (SD 1.0). The overall median MMSE score was 27 (IQR 25–29), the median grip strength for females was 17 kg (IQR 14–20), and 28 kg (IQR 23–33) for male participants.In In n = 2 317) of the study population, with 2.9% (n = 68) missing. In total, 994 (42.9%) participants had a high composite fitness score, and 694 (30.0%) had a low composite fitness score. The remaining 629 (n = 27.1%) participants did not meet the criteria of the high or low composite fitness score. The characteristics of the participants stratified by composite fitness score are shown in n = 421 deaths [60.7%]), and had shorter survival time (median 1 400 days [IQR 661–1 826]) compared to the participants with a high composite fitness score .The composite fitness score was calculated for 97.1%  of the participants died. Consistent in all cohorts, there was an inverse association between LDL-C and 5-year mortality risk, pooled HR 0.87 (95% CI: 0.80–0.94). Within the cohorts, the HR ranged from 0.76 (95% CI: 0.59–0.99) in the LiLACS-NZ non-Māori cohort, to HR 0.94 (95% CI: 0.69–1.29) in the LiLACS-NZ Māori cohort, as shown in p = .98), while in the groups with low functional ability low LDL-C was associated with a decreased risk of mortality . In line with these results, there was no association between LDL-C and mortality in strata with high cognitive function , while in the group with low cognitive function scores low LDL-C was associated with a decreased risk of mortality . Results were mostly consistent across the cohorts .In  cohorts . Moreovep = .78), while in participants with a low-composite fitness score, a 1 mmol/L increase in LDL-C was associated with a lower mortality risk .The forest plot in The association between LDL-C and mortality was similar in participants with and without ACVD. Also, the effect of the composite fitness score on the association did not materially change when we additionally stratified on lipid lowering treatment . FindingIn this 2-stage IPD meta-analysis, there was an inverse association between baseline LDL-C and all-cause mortality, which was most pronounced in participants with a low-composite fitness score. This trend of effect modification by the composite fitness score was similar for participants with and without lipid-lowering medication.Within the BELFRAIL study  it was eResults of this study confirm that in very old adults lower LDL-C is associated with higher mortality risk, and the observed trend of effect modification by the composite fitness score suggests that fitness may account for some of the variation in the association that was seen by others . These fA key strength of this study is that it combined individual data of 5 substantial cohorts of very old adults from different geographical locations, and diverse cultures, adding to the generalizability of the results. In addition, the 2-stage IPD meta-analysis design allowed us to take into account unmeasured differences between the cohorts  and to combine the data despite methodologic heterogeneity between the studies.There are some limitations that have to be considered when interpreting the results of this study. First, we performed analyses on observational data, therefore no causal inference can be made. Therefore, our results should not be interpreted as evidence that in some people lowering LDL-C increases mortality risk. One could hypothesize that the found association is the reflection of an epiphenomenon where a possibly genetic pathophysiological abnormality causes an increased mortality risk and decrease in levels of LDL-C in parallel for some older people . AlternaIn this very old population, there was an inverse association between baseline LDL-C and all-cause mortality, which was most pronounced in participants with a low-composite fitness score. The results of this observational study add a small piece of evidence to unravel the complex relationship between LDL-C and mortality in older adults. More research is needed to investigate important questions on the underlying causal mechanisms and clinical and ethnic-specific implications.glad148_suppl_Supplementary_FileClick here for additional data file.


Brassica rapa is a widespread naturalized weed that is found throughout temperate North and South America, and it is a frequent weed among winter cereal crops in Argentina and in Mexico. Broadleaf weed control is based on glyphosate that is used prior to sowing and sulfonylureas or mimic auxin herbicides that are used once the weeds have already emerged. This study was aimed at determining whether a convergent phenotypic adaptation to multiple herbicides had occurred in B. rapa populations from Mexico and Argentina by comparing the herbicide sensitivity to inhibitors of the acetolactate synthase (ALS), 5-enolpyruvylshikimate-3-phosphate (EPSPS), and auxin mimics. Five B. rapa populations were analyzed from seeds collected in wheat fields in Argentina (Ar1 and Ar2) and barley fields in Mexico . Mx1, Mx2, and Ar1 populations presented multiple resistance to ALS- and EPSPS-inhibitors and to auxin mimics , while the Ar2 population showed resistance only to ALS-inhibitors and glyphosate. Resistance factors ranged from 947 to 4069 for tribenuron-methyl, from 1.5 to 9.4 for 2,4-D, and from 2.7 to 42 for glyphosate. These were consistent with ALS activity, ethylene production, and shikimate accumulation analyses in response to tribenuron-methyl, 2,4-D, and glyphosate, respectively. These results fully support the evolution of the multiple- and cross-herbicide resistance to glyphosate, ALS-inhibitors, and auxinic herbicides in B. rapa populations from Mexico and Argentina.Herbicide-resistant weeds have been identified and recorded on every continent where croplands are available. Despite the diversity of weed communities, it is of interest how selection has led to the same consequences in distant regions.  Weeds evolve and adapt to the environment in response to management practices ,2. HerbiThe overreliance on glyphosate across vast regions with a low diversity of control strategies explains the increase in glyphosate-resistant cases ,8. DespiBrassica rapa in Argentina occurred through gene flow and introgression between feral populations of glyphosate-resistant transgenic B. napus (oilseed rape), imidazolinone-tolerant non-transgenic cultivars, and wild B. rapa, where the persistence and spread of resistance were facilitated by the high selection pressure imposed by glyphosate and acetolactate synthase (ALS)-inhibiting herbicides  was added. The samples were incubated at 25 °C for 90 min. Next, a 500 µL solution of sodium hydroxide (0.6 N NaOH) and sodium sulfite (0.22 N Na2SO3) [1:1 (v/v)] was added and mixed. The absorbance was measured using a spectrophotometer mod. DU-640  at 380 nm. The results were expressed as micrograms of shikimate per milliliter HCL solution (µg shikimate mL−1) using a calibration curve with known concentrations of shikimate . The experiment was conducted in a completely randomized design with five tissue samples from each population and glyphosate dose. The experiment was replicated twice.Shikimic acid accumulation was determined in plants with four leaves and treated with glyphosate  were calculated, and the accuracy of the models was analyzed [The data of the dose–response experiments were used to build non-linear log-logistic regression models with three parameters . GR50, Lanalyzed .B. rapa populations. When necessary (p < 0.05), the means were distinguished using the Tukey test .ANOVAs were performed to assess the differences between the ethylene production, shikimic acid accumulation, and ALS activity in the


The crosslinked (PAA-co-PAM)-DPNR was prepared via emulsion graft copolymerization in the presence of MBA as a crosslinking agent. The modified DPNR was mixed with various contents of silica (10 to 30 phr) to form the composites. The existence of crosslinked (PAA-co-PAM) after modification provided a water adsorption ability to DPNR. The swelling degree values of composites were found in the range of 2217.3 ± 182.0 to 8132.3 ± 483.8%. The addition of silica in the composites resulted in an improvement in mechanical properties. The crosslinked (PAA-co-PAM)-DPNR with 20 phr of silica increased its compressive strength and compressive modulus by 1.61 and 1.55 times compared to the unloaded silica sample, respectively. There was no breakage of samples after 80% compression strain. Potassium nitrate, a model fertilizer, was loaded into chitosan beads with a loading percentage of 40.55 ± 1.03% and then coated with the modified natural rubber/silica composites. The crosslinked (PAA-co-PAM)-DPNR/silica composites as the outer layers had the ability of holding water in their structure and retarded the release of fertilizer. These composites could be promising materials for controlled release and water retention that would have potential for agricultural application.The crosslinked poly(acrylic acid- The controlled release system is an effective strategy for various applications such as biomedical ,2 and agHevea Brasiliensis rubber trees. NR has high elasticity and film-forming ability. The excellent properties of NR make it suitable for coating substances [Bio-based polymeric materials are interesting and have advantages for several fields including agricultural application because of their low cost, low toxicity and environmental friendliness. Natural rubber (NR) is a biopolymer, obtained from bstances . NR can bstances ,10. It cco-polyacrylamide) is a copolymer that possesses hydrophilic properties with a large number of polar groups [co-polyacrylamide)-based superabsorbent materials through the graft copolymerization of okara with acrylic acid and acrylamide [co-polyacrylamide)-based materials were used as soil supplements for plant growth application under a water-limited condition.Superabsorbent polymer materials are promising materials for the agricultural sector. They are composed of a three-dimensional polymeric network structure that can absorb and hold water and nutrients during the growth period. Poly(acrylic acid-r groups . J. Zhu rylamide . They exco-acrylamide)-grafted deproteinized natural rubber ((PAA-co-PAM)-DPNR) was successfully prepared via emulsion graft copolymerization which was a water-based process [co-PAM was grafted on the particle surface of natural rubber. The monomer contents were varied at 10 and 30 phr. The grafting efficiency and grafting percentage were found in the range of 20.8–38.9 and 2.1–9.9%, respectively. However, when the products were used in the wet condition, the ungrafted polymers could be dissolved in the aqueous medium. So, the undesired performance in application was achieved. Moreover, the modified natural rubber was then mixed with silica. The modified natural rubber/silica composites showed enhanced mechanical properties due to strong interactions between the silica and modified natural rubber. Natural rubber/filler composites have been studied for improving the properties of natural-rubber-based materials, including mechanical and thermal properties to promote application efficacy meaning they can be used in a wide range of applications. The silica-reinforced natural rubber composites were used for many rubber applications such as tire manufacturing [w/v). Therefore, the natural rubber/silica-based materials could be applied for use in various coating applications such as biomedical devices, microelectronics and the automobile industries. P. Boonying et al. prepared the biocomposite coating films from lignin and natural rubber grafted with polyacrylamide (NR-g-PAM) for slow release fertilizer [g-PAM acts as the compatibilizer to enhance the stability of lignin/NR dispersions. It can enhance the hydrophilicity to the composites and allow water transport through the lignin/NR composite film. The Li/NR-g-PAM-coated urea revealed a slow release of N, but only 60% of the total N after 112 days. Therefore, the natural rubber/filler composites could have potential for various applications.According to the our previous work, poly-DPNR was prepared using N’,N’-Methylenebisacrylamide (MBA) as a crosslinking agent. The crosslinked (PAA-co-PAM)-DPNR latex was then mixed with silica to prepare the natural rubber/silica composites and applied as coating materials for fertilizer. The crosslinked (PAA-co-PAM)-DPNR/silica composites were characterized and coated on chitosan beads. The release behavior and water retention ability of the crosslinked (PAA-co-PAM)-DPNR/silica-covered chitosan beads were investigated. In this work, to prepare the natural-rubber-based composites for coating fertilizer, the deproteinized natural rubber was modified via emulsion graft copolymerization with acrylic acid and acrylamide. In order to enhance the grafting of PAA-3) was obtained from Kemaus . Deionized (DI) water was used throughout the study. Natural rubber latex preserved with high ammonia  was purchased from Chemical and Materials Co., Ltd. . Acrylic acid (AA) and cumene hydroperoxide (CHP) were obtained from Aldrich . The purification of the AA monomer was performed by passing through a column packed with alumina adsorbent before polymerization . AcrylamThe crosslinked (PAA-co-PAM)-DPNR via emulsion graft copolymerization was performed according to a previous study with some modifications . The depTo determine the monomer conversion, the dried samples were weighed before and after immersion in ethanol for 24 h. The samples were then dried at 60 °C for 24 h. The monomer conversion was calculated as follows :(1)Monomco-PAM was removed via extraction with DI water. The dried samples were immersed with DI water for 72 h. The medium was changed every 8 h. The samples were then dried at 60 °C for 24 h. The grafting efficiency and grafting percentage were calculated as follows [For the calculation of grafting efficiency and grafting percentage, the ungrafted PAA- follows :(2)GraftThe morphology of samples was studied using transmission electron microscopy (TEM). A drop of diluted latex was put onto a carbon-coated copper grid. The sample was stained with osmium tetroxide in the carbon–carbon double bonds of natural rubber for 24 h to increase the contrast . The morThe dried samples were weighed and then immersed in DI water to extract the sol fraction from the matrix at room temperature for 72 h. The medium was changed 3 times a day. The samples were subsequently freeze-dried overnight. The samples were weighed after drying and the gel fraction was calculated as follows :(4)Gel fco-PAM)-DPNR/silica composites were prepared by mixing the crosslinked (PAA-co-PAM)-DPNR latex with different silica contents. The silica was prepared via the precipitation method by treatment with rice husk ash with 1 M of HCl solution for 3 h to remove the metallic oxide. The ash was then filtered and washed with DI water until the pH was neutral. The purified ash was dried in the hot air oven at 110 °C for 12 h. The dried sample was added into 1 M of NaOH solution and stirred for 12 h at 90 °C to obtain the sodium silicate. The undissolved product was removed via filtration. To prepare the silica, 1 M of acetic acid was dropped into the sodium silicate solution until the pH was neutral under stirring at room temperature. The precipitate silica was obtained and then separated via filtration followed by washing with the excess of DI water. The resulting silica was dispersed in DI water with the solid content of 3%. The silica dispersion was dropped into the latex and stirred at 100 rpm for 3 h at room temperature. The silica contents were varied as 10, 20 and 30 phr. The chemical compositions are shown at The crosslinked (PAA-co-PAM)-DPNR/silica composites using a Tensor 27 FTIR spectrometer . The scanning of each spectrum was performed with 64 scans at a resolution of 4 cm−1. The FTIR spectra of all samples were recorded in the range of 4000–400 cm−1.Fourier-transform infrared spectroscopy (FTIR) with an attenuated total reflection (ATR) mode was used to study the chemical structure of the crosslinked (PAA-co-PAM)-DPNR/silica composites were determined via energy-dispersive spectroscopy coupled with SEM (SEM/EDS).The morphology of samples was observed using a scanning electron microscope (SEM). The samples were frozen and broken in liquid nitrogen. The samples were fixed on the stub using conductive carbon tape and kept in a desiccator overnight. The samples were sputter-coated with gold under a vacuum for 3 min. The cross-section of samples was observed through a JSM-6010LV . Moreover, the element compositions of the crosslinked SwellThe contact angle measurement was carried out by dropping water via a microsyringe on the rubber sample. After 20 s, the water droplet on the surface was recorded and the angle formed between the interface was measured using ImageJ software .The thermal properties of the composites were traced via thermogravimetric analysis using a TGA/DSC1 . A total of 10 mg of the dried sample was added in a sample pan. The pan without the addition of the sample was used as a reference. The reference and sample pans were placed into the furnace. The measurement was performed in the temperature range of 50 to 600 °C at a heating rate of 10 °C/min under nitrogen atmosphere.co-PAM)-DPNR/silica composites were determined using a TA.XT plus texture analyzer . The sample was immersed in DI water before measurement and then cut into a cylindrical shape with a diameter of approximately 10 mm and a thickness of 2 mm. The swollen sample was compressed to 80% strain with a fixed strain rate at 0.05 mm/s at room temperature. The compressive modulus was determined from the slope of the stress–strain curve (5–10% strain) [The compressive properties of the crosslinked (PAA- strain) . The meaw/w of chitosan (CS) solution in acetic acid (1% v/v) was dropped into 50 mL of sodium tripolyphosphate (TPP) solution using a syringe with a needle. The concentration of TPP was kept constant at 1% w/v. The sample was continuously stirred at 200 rpm for 4 h. The CS beads were removed from the solution and washed with DI water. The beads were then dried at 35 °C for 24 h. The crosslinked (PAA-co-PAM)-DPNR/silica latex with various silica contents was dropped on CS beads. The weight ratio of crosslinked (PAA-co-PAM)-DPNR/silica and CS was kept constant at 1:15. The sample was dried at 60 °C for 24 h to obtain crosslinked (PAA-co-PAM)-DPNR/silica-coated CS beads.The chitosan beads were prepared following the ionic gelation technique according to J.J. Perez et al. . The 10 co-PAM)-DPNR/silica-coated CS beads were immersed in DI water for 1 h. The samples were subsequently freeze-dried overnight. The freeze-dried beads were fixed on a stub using conductive carbon tape. Then, they were sputter-coated with gold under vacuum for 3 min. The samples were subjected to FEI Quanta 450 SEM .The morphology of the prepared beads was observed using a scanning electron microscope (SEM). Before SEM observation, the crosslinked (PAA-The water retention test was performed by measuring the change in the remaining weight of water in the sample container. The sample container was filled with the sample and sand (Wo). Sand with a size in the range of 425–625 µm determined via passing it through a sieve mesh was dried at 105 °C for 24 h before testing. The different types of beads (5% w) were buried in 30 g of sand in a plastic cup, followed by the addition of DI water (30% w) (Ws). The weight of the sample container was recorded at certain time intervals (Wt). The measurement was performed at 25 and 45 °C. The water retention was calculated as follows ,29:(6)WaTo encapsulate potassium nitrate in the CS beads, the dried CS beads were immersed in 20% w of potassium nitrate solution for 4 h. Subsequently, the samples were dried at 35 °C for 24 h . The loaThe release experiment was performed by placing potassium-nitrate-loaded beads in a dialysis bag  and then immersed in 100 mL of DI water. The test was performed at 25 °C. The released amount of potassium nitrate was determined by measuring the conductivity of aqueous medium at various time intervals.co-PAM)-DPNR was prepared via graft copolymerization of a comonomer of acrylic acid and acrylamide. The MBA crosslinking agent was introduced during the polymerization process and its contents were varied at 0.25 and 0.50 by weight percentage of the comonomer, which were noted as M0.25/P30-DPNR and M0.50/P30-DPNR, respectively. The added crosslinking agent can link the PAA-co-PAM chains and form a crosslinked network of hydrophilic polymers into the natural-rubber-based matrix as the potential materials for application [co-PAM)-DPNR with different MBA contents were compared to the uncrosslinked sample (P30-DPNR). From the results shown in co-PAM molecular chains and can link the ungrafted polymers to the natural-rubber-based structure. This resulted in an increase in the grafting efficiency and grafting percentage of the crosslinked samples.The MBA-crosslinked (PAA-co-PAM)-DPNR particles. From the TEM images, the particles exhibited a core–shell morphology. All particles showed the dark color particles of DPNR covered with the (PAA-co-PAM) shell [co-PAM after modification. After adding the MBA crosslinking agent, it could be clearly seen that at 0.50% w of MBA, the formation of linkage around the rubber surface occurred, suggesting that the network structure took place between rubber particles [co-PAM can be formed and covered on the natural rubber surface without being linked by chemical bonds, as seen in co-PAM during the chain propagation [co-PAM chains could have resulted from and also be associated with ungrafted chains. Nevertheless, the addition of a high amount of crosslinking agent not only chemically bonded with PAA-co-PAM chains on the individual natural rubber particles, but it might have also led to crosslinking them on the other rubber particles.M) shell . This coarticles . This caarticles , the frepagation . Therefoco-PAM) chains in their structure. From co-PAM)-DPNR was higher than that of the uncrosslinked sample. The gel fraction of P30-DPNR was found to be 78.8 ± 1.3%. When the MBA crosslinking agent was introduced, the gel fraction increased to 80.6 ± 0.2 and 88.6 ± 0.8% for M0.25/P30-DPNR and M0.50/P30-DPNR, respectively. It can be seen that the addition of a higher crosslinking agent leads to an increase in the gel fraction due to a larger crosslinking site in the sample. Indeed, natural rubber is a hydrophobic molecule composed of polyisoprene chains, while the PAA-co-PAM is a hydrophilic polymer. When the samples are immersed in aqueous medium, the ungrafted PAA-co-PAM segments can dissolve in aqueous medium, resulting in a reduction in the gel fraction. The crosslinking agent interacted with PAA-co-PAM chains and linked them to a rubber-based structure via chemical bonds. Thus, it was suggested that the presence of an MBA crosslinking agent can improve structural stability and hold the hydrophilic units on the sample structure. This would bring advantages for use in application in terms of water adsorption ability [The gel fraction of the modified natural rubber was studied to investigate the ability of samples for holding the -grafted deproteinized natural rubber/silica composites were prepared to be used as coating materials for fertilizer. From the above section, the crosslinked (PAA-co-PAM)-grafted DPNR was firstly prepared via emulsion graft copolymerization using the CHP/TEPA redox initiator system in the presence of MBA as a crosslinking agent. The crosslinked (PAA-co-PAM)-DPNR latex was then mixed with silica via the wet mixing process to produce natural rubber/silica composites, as shown in The crosslinked (PAA-co-PAM)-DPNR/silica composites were compared to DPNR, M0.50/P30-DPNR and silica. The characteristic peaks of DPNR appeared at 1664, 1446, 1374 and 841 cm−1, which were assigned to the vibration bands of C=C, -CH2, -CH3 and =CH, respectively [−1 (OH stretching), 3206 cm−1 (NH stretching), 1663 cm−1 (C=O stretching), 1613 cm−1 (NH bending), 1563 cm−1 (-COO−) and 1240 cm−1 (C-O stretching) were observed [−1 (Si-O-Si asymmetric stretching), 963 cm−1 (Si-OH bending), 794 cm−1 (Si-O symmetric stretching) and 458 cm−1 (Si-O bending) [co-PAM)-DPNR/silica composites, both characteristic peaks of M0.50/P30-DPNR and silica appeared. However, the peaks of OH stretching and NH stretching were shifted to 3350 cm−1 and 3204 cm−1, respectively. The peaks of Si-OH and Si-O-Si were shifted from 963 to 974 cm−1 and 1065 to 1080 cm−1, respectively. These demonstrated the generation of H-bonding between modified natural rubber and silica and confirmed the incorporation of silica in the composites [−1 of Si-O-Si stretching compared to those at 1374 cm−1 of -CH3 stretching are shown in The FTIR was used to characterize the chemical structure and functional groups of the prepared samples, as shown in observed . These cbending) . In the co-PAM)-DPNR/silica composites compared to the sample without the addition of silica. As seen in co-PAM in natural-rubber-based materials can promote the water adsorption capacity due to crosslink network structure formation, together with polar functional groups such as carboxylic acid and amide groups presenting in the chemical structure of PAA-co-PAM for the highly effective absorption. When the silica was added, the composites showed a lower swelling degree than that of MBA0.50/P30-DPNR. The swelling degree of M0.50/P30-DPNR/Si10, M0.50/P30-DPNR/Si20 and M0.50/P30-DPNR/Si30 were 8132.3 ± 483.8, 5232.1 ± 435.6 and 2217.3 ± 182.0%, respectively. The swelling degree decreased when silica increased. This could be explained by the fact that since the polar functional groups of PAA-co-PAM play an important role in the adsorption process, these groups are strongly interacted with silica and obtain more compact structures. This might lead to an increase in the crosslink density in the composites. A higher crosslink density results in a lower swelling degree, as can be observed in other research works [The swelling degree and characteristics of the composites after immersion in water were determined to estimate the water absorption capacity of the composites, as displayed in ch works . In addico-PAM after modification can enhance the hydrophilicity to the natural-rubber-based structure. For the M0.50/P30-DPNR/silica composites, their contact angle values were higher than those of M0.50/P30-DPNR and tended to increase with an increase in silica contents. The contact angle values of M0.50/P30-DPNR/Si10, M0.50/P30-DPNR/Si20 and M0.50/P30-DPNR/Si30 were found to be 45.8°, 65.2° and 69.1°, respectively. These results were corresponded to the swelling experiment. The composites with more interaction between polar groups of PAA-co-PAM and silica were more hydrophobic and showed a lower swelling degree. However, their contact angle values were lower than that of DPNR, suggesting that they still had hydrophilic behavior in their structure.co-PAM chains [co-PAM decomposed at a temperature between 336 and 475 °C. From thermograms, the temperature at 10% of weight loss (T10), temperature at maximum process rate (Tmax) and residue of the degradation process are reported in 10 and Tmax values increased when the silica contents increased. Similar results have been reported in other research works [10 and Tmax to a higher temperature demonstrated that the composites had stability over wide temperature ranges. It could be indicated that the thermal stability of the composites was improved because the silica particles dispersed in the natural rubber matrix could adsorb heat energy and retard the heat transfer to natural rubber [The TGA and DTG thermograms of M0.50/P30-DPNR and M0.50/P30-DPNR/silica composites with various silica contents are displayed in M chains . The DPNch works . The shil rubber . MoreoveThe mechanical properties of the composites are also important characteristics for the application. Since composites are used as coating materials for fertilizer in agricultural applications, they might be soaked in water and buried in the soil. Therefore, the compressive properties of the composites were determined in the condition of swollen samples with water. co-PAM)-DPNR/silica-coated CS beads in the dry state is shown in co-PAM)-DPNR/silica-coated CS beads with various silica contents, the size was higher than that of neat CS beads due to the coverage of high water-absorbing materials on CS beads. Their size was found to be in the range of 1246.2 ± 108.1 to 1398.9 ± 70.4 µm. Since the coating materials on CS beads exhibited high water absorption capacity, the porous morphology appeared on their surface. The pores on the surface of beads are formed via the sublimation of trapped water within the crosslinked network structure of the modified natural rubber/silica composite film after freeze drying [The CS beads were prepared via the ionic gelation method. The different types of composites were used to coat onto CS beads. The physical appearance of crosslinked  was determined because this pH was in the range of the optimal pH (5.5 to 6.5) for the growth of most plants such as ginger, cassava, maize, wheat, French bean and tomato [3 from CS reached 47.40 ± 1.37% after 2 days. Then, it reached 79.41 ± 2.01% after 14 days. The delayed release rate of KNO3 was observed when the beads were coated with the prepared natural-rubber-based composites. The release percentages of KNO3 from modified-natural-rubber/silica-coated CS beads were lower than those of the uncoated sample in all studied time periods. Thus, the coating with natural rubber/silica composites provided the controlled release of KNO3 in aqueous medium. The coating agent acted as a protective layer against the fast release of fertilizer [2). According to this model, the equation is Qt = ktn, where Qt is the fraction of fertilizer release at time t, k is the release rate constant and n is the release exponent for indicating the characteristics of the release mechanism. For n < 0.5, the transport mechanism followed Fickian diffusion, whereby the diffusion is the main release mechanism. In the case of n > 1, the transport mechanism is classified as Super Case II transport, in which the nutrient transport mechanism is associated with the relaxation process of hydrophilic polymers upon swelling in water. From this result, 0.5 < n < 1 was obtained, indicating a non-Fickian transport release mechanism. This demonstrated that the release occurred through both processes [3. The stronger interactions between the crosslinked (PAA-co-PAM)-DPNR and silica became more hydrophobic, a characteristic that provided the stronger barrier to prevent the release of KNO3 [3 can be encapsulated in CS beads and the crosslinked (PAA-co-PAM)-DPNR/silica composites, as the outer layers allow the water and dissolved substances to pass through their network structure with a low diffusion rate. Therefore, these materials can retard the release and enhance the water holding capacity that would have potential for applications [The loading capacity of the KNOJ. Perez . Then, td tomato . The ferrtilizer . After 2rocesses . The coa of KNO3 . This reco-PAM)-DPNR was successfully prepared via emulsion graft copolymerization in the presence of MBA as a crosslinking agent. The grafting efficiency and grafting percentage were increased from 38.9 ± 2.1 to 81.5 ± 3.5% and from 9.9 ± 1.7 to 24.4 ± 1.1% when MBA was increased from 0 to 0.50% w of the monomer, respectively. The addition of a crosslinking agent can hold the hydrophilic PAA-co-PAM chains to a natural-rubber-based material and form a network structure that has the ability of the absorption of water. The crosslinked (PAA-co-PAM)-DPNR was mixed with silica to prepare natural rubber/silica composites. Due to the strong interaction between polar groups of the crosslinked (PAA-co-PAM)-DPNR and silica, the composites became more hydrophobic, as determined via contact angle measurement. The swelling degree in the water of crosslinked (PAA-co-PAM)-DPNR/silica composites was found in the range of 2217.3 ± 182.0 to 8132.3 ± 483.8%, when the silica was added at 10 to 30 phr. The presence of silica was found to improve the mechanical properties of the composites. For the crosslinked (PAA-co-PAM)-DPNR incorporated with 20 phr of silica, its compressive strength and compressive modulus increased by 1.61 and 1.55 times those of the unloaded silica sample, respectively. The composites exhibited good structural stability without the breakage of samples after immersion in water and compression. These composites were employed as coating materials for fertilizer. The crosslinked (PAA-co-PAM)-DPNR/silica composites were coated on potassium-nitrate-loaded chitosan beads. They had the ability of retaining water molecules and exhibited slower potassium nitrate release. Therefore, these composites had the ability for water adsorption, water retention and controlled release behavior that would be useful for agricultural application.The crosslinked (PAA-


Background: The presence of actionable alterations in advanced biliary tract cancer patients opened new therapeutic possibilities for second-line treatments. However, for around 60% of the patients, chemotherapy remains the only therapeutic option. The aim of our study was to evaluate outcomes and prognostic parameters in patients with intrahepatic cholangiocarcinomas treated with second-line chemotherapy. Methods: A total of 255 consecutive metastatic intrahepatic cholangiocarcinoma (ICC) patients were retrospectively reviewed and clinicopathologic and survival data were collected. Results: Fourty-four percent of ICC patients underwent second-line chemotherapy. In particular, younger ICC patients with better ECOG PS status, and with disease control after first-line chemotherapy were those who were treated with second-line treatments. Median progression-free survival in the patients treated with second-line chemotherapy was 3 months. Finally, the patients affected by intrahepatic cholangiocarcinoma with better ECOG PS, with prior surgical resection of the primary tumor, who responded to first-line chemotherapy, and had better progression-free survival with second-line chemotherapy, were associated with better outcomes in multivariate analysis. Conclusions: Not all patients seem to benefit from second-line chemotherapy. To improve therapeutic decisions, performance status and disease control with first-line chemotherapy should lead to the decision on the usefulness of second-line treatments in advanced ICC patients. Biliary tract cancers (BTCs) are rare tumors that account for less than 1% of all cancers worldwide, even though both incidence and mortality have risen in the last decades ,3,4,5. TBecause aBTC were found to be enriched in genomic alterations, several trials have been exploring the potential role in the second-line settings of targeted therapies . A s2 and cisplatin at 25 mg/m2, each on days 1 and 8 of a 21-day regimen) or gemcitabine and oxaliplatin . In the case of unacceptable toxicities after platinum-based chemotherapy doublet, monotherapy without platinum derived was continued until disease progression when second-line chemotherapy was introduced. The main regimens used for second-line chemotherapy were 5-fluoropyrimidine-based doublets such as XELOX/FOLFOX, XELIRI/FOLFIRI, or capecitabine/De Gramont regimen. The regimens were as follows: FOLFOX  plus oxaliplatin at 100 mg/m2 on day 1 every 2 weeks), XELOX , FOLFIRI  plus irinotecan at 180 mg/m2 on day 1), XELIRI (capecitabine at 2000 mg/m2/day on days 1–14 and irinotecan at 200 mg/m2 on day 1 every 3 weeks), capecitabine (2000 mg/m2/day on days 1–14 every 3 weeks) and De Gramont regimen . All patients were evaluated with computed tomography (CT) or magnetic resonance scan every 3 months during first-line and second-line treatment. According to the RECIST evaluation (https://recist.eortc.org/ accesses on 15 July 2023), tumor response was classified as a progressive disease, stable disease, or complete or partial remission, corresponding to the evaluation criteria in solid tumors . Baseline clinical and pathological parameters were recorded prior to palliative chemotherapy start. PFS is defined as the time period from beginning first-line chemotherapy (PFS1) or second-line chemotherapy (PFS2) until disease progression or death. We documented patients’ outcomes , as well as Best Response to first-line chemotherapy and the type of second-line treatment.This study protocol was reviewed and approved by the Ethics Committee “Area Vasta Emilia Nord” (protocol 78/2017/O/OSSN), which was in accordance with the Declaration of Helsinki and Good Clinical Practice. It was conducted in three Italian Hospitals . A total of 255 patient files were retrieved using electronic medical records. All consecutive patients with histologically proven metastatic intrahepatic cholangiocarcinomas treated with first-line and second-line palliative chemotherapy between January 2012 and December 2018 were eligible for this study. The biliary tract cancers consisting of intrahepatic cholangiocarcinomas, extrahepatic cholangiocarcinoma, gallbladder, or ampullary cancers were excluded from this study. The patients were treated with first-line chemotherapy until disease progression or unacceptable toxicity. The main regimen for the first-line treatment was the combination of gemcitabine and cisplatin  were included in multivariate Cox regression analysis. IBM SPSS Statistics v 25.0 (IBM) was used to perform all computational analyses. The p-value was bilaterally tested, and values less than 0.05 were regarded as statistically significant. Clinical and pathologic, as well as long-term survival data, were collected and reviewed. OS from metastatic disease was calculated from the date of metastatic ICC diagnosis to the date of death. PFS after first-line and second-line chemotherapy (PFS1 and PFS2) were defined as the time of first-line or second-line therapy initiation to disease progression or death. Best Response to first-line therapy was divided into progressive and non-progressive disease . OS, PFS1, and PFS2 curves were compared by log-rank test and transcribed into Kaplan-Meier diagrams. OS for metastatic disease is defined as the time period from the application of first-line palliative chemotherapy until death. Schematic view of PFS1 and PFS2 are shown in Out of 255 consecutive metastatic ICC patients included in our study, 143 (55.4%) were males and 115 (44.6%) were females. The median age at diagnosis was 66.5 years (range 34–86 years). Fourty-four percent (113/255) of metastatic ICC patients underwent second-line chemotherapy. The median of PFS2 in patients treated with second-line chemotherapy was 3 months (range 1–30 months). In the whole group, the median OS from metastatic disease occurrence was 11 months (range 1–1471 months).The basic clinical and pathological characteristics of the 255 ICC patients are detailed in p < 0.001), as demonstrated in p < 0.001), as demonstrated in p = 0.002) as demonstrated in We divided patients into two groups according to PFS1: with PFS1 less or more than the median. ICC patients with a shorter median PFS1 have a worse median OS  compared to ICC patients with a longer than median PFS1 , with a statistically significant difference (p = 0.93).We evaluated the type of second-line chemotherapy treatment (fluoropyrimidine-based or gemcitabine-based). The majority of the patients (66%) were treated with fluoropyrimidine-based chemotherapy, and the rest were treated with gemcitabine-based chemotherapy. Interestingly, no statistically significant differences were seen between the two groups according to the type of second-line chemotherapy  . We added all of these variables to the multivariate analysis. For the Cox analysis, the following reference groups were selected: the median age less than the median, male sex, ECOG PS 0–1 group, no surgery group, median PFS1 less than the median, Best Response to first-line chemotherapy with disease control, gemcitabine-based second-line chemotherapy, and median PFS2 less than the median. The Cox proportional hazards model analysis showed that no surgery and a median PFS2 less than the median were associated with a significantly higher HR for death . Younger age was also associated with a higher HR for death, with a tendency for a statistically significant difference . In contrast, ECOG PS 0–1 group and Best Response to first-line chemotherapy with no progressive disease were associated with a significantly lower HR for death , see To outline the clinically relevant factors associated with predicting the impact on OS, we used univariate and multivariate COX regression models . Univari= 0.004) . On the In our series, 44% of ICC patients were treated with second-line chemotherapy. In particular, younger ICC patients with better ECOG PS status, with disease control after first-line chemotherapy, were those who underwent second-line treatments. Further, the ICC patients with better ECOG PS with prior surgical resection of the primary tumor who responded to first-line chemotherapy and had better PFS2 were associated with a better outcome in multivariate analysis.p < 0.001). The median PFS was 8.0 months in the cisplatin–gemcitabine group and 5.0 months in the gemcitabine-only group (p < 0.001). In addition, the response rate among patients treated with doubled chemotherapy was higher compared to the patients’ group treated with gemcitabine in monotherapy  [Even if the disease were rare, the incidence of ICC has demonstrated an increasing tendency in recent decades. According to some authors, since the early 1990s, there has been a 14% increase in incidence per year . The pro= 0.049) . Cholang= 0.049) ,23,24. p = 0.031). The results of the ABC-06 clinical trial demonstrated that FOLFOX chemotherapy can improve the OS in patients with good ECOG PS with aBTC who have been previously treated with cisplatin and gemcitabine. Therefore, in Western countries, FOLFOX became a standard of care in second-line settings [p = 0.031) [p = 0.004) [Until 2019, there was no recommended regimen in the second-line setting. It has been estimated that approximately 15–25% of aBTC receive second-line therapy due to rapidly worsening performance status after first-line treatment ,14,25. Ip = 0.65). Similar findings were observed in the Italian cohort (174 patients). The authors concluded that fluoropyrimidine monotherapy is as active as fluoropyrimidine doublets in aBTC in second-line, regardless of the patient performance status and country, and could be a therapeutic option in this setting [p = 0.009), although the PFS (p = 0.43) and OS (p = 0.88) did not significantly differ between these groups. Kim et al. concluded that fluoropyrimidine–platinum combination therapy was not associated with improved survival outcomes, as compared with fluoropyrimidine monotherapy [The comparison with fluoropyrimidine monotherapy was not available in the ABC-06 trial. Several authors suggest that fluoropyrimidine monotherapy is as active as fluoropyrimidine doublets in aBTC in second-line settings ,28. The  setting . A simil setting . The autotherapy .p = 0.140) and PFS  was observed, but these differences did not reach statistical significance. Of note, contrary to previous studies, they reported prolonged OS with combination chemotherapy compared to single-agent .Regarding the reported efficacy of second-line chemotherapy, the median PFS in ICC patients who received second-line treatment in our study was 3 months. Similar results were described by Lamarca et al. who evaluated 761 aBTC patients . This syPatients with aBTC treated with second-line chemotherapy display prognostic heterogeneity ,27,28,30Our study is limited by its retrospective nature for data collection and the high rate of missing data for biological data, though the evaluated ICC population is more homogeneous compared to other studies which included all aBTC. Moreover, another limitation of our study was that routine molecular profiling was not available for all patients participating in our study because it was not the standard of care at the time of the study’s conduct; thus, the impact on patients with specific molecular aberrations is not known . NowadayIn conclusion, in our study, we evaluated only ICC patients, a more homogeneous group compared to most studies that evaluated all aBTC . In our series, approximately 44% of ICC patients underwent second-line palliative treatment. Not all patients seem to benefit from second-line chemotherapy and it has to be evaluated on an individual basis because the prognosis remains poor. No particular regimen seems to be superior to others in our series, and this underlines the need for new treatments. To improve therapeutic decisions, performance status and disease control with first-line chemotherapy should lead the decision on the usefulness of second-line treatments in advanced ICC patients, though randomized clinical trials are needed to confirm this.


IntroductionDuring the coronavirus disease 2019 (COVID-19) pandemic, a rapid and significant transformation in patient management occurred across the healthcare system in order to mitigate the spread of the disease and address resource constraints. Numerous surgical cases were either postponed or canceled, permitting only the most critical and emergent cases to proceed. The impact of these modifications on patient outcomes remains uncertain. The purpose of this study was to compare time-to-surgery and outcomes of open reduction and internal fixation for trimalleolar ankle fractures during the pandemic to a pre-pandemic group. We hypothesized that the pandemic group would have a prolonged time-to-surgery and worse outcomes compared to the pre-pandemic cohort.Materials and methodsThis retrospective cohort study was conducted within a single healthcare system, examining the treatment of trimalleolar ankle fractures during two distinct periods: April to July 2020 (COVID-19 group) and January to December 2018 (2018 group). Cases were identified using Current Procedural Terminology code 27822. Information on demographics, fracture characteristics, and outcomes was obtained through chart review. Outcomes analyzed included time-to-surgery, mean visual analog scale scores, ankle strength and range of motion, and complications.ResultsCOVID-19 and 2018 groups consisted of 32 and 100 patients, respectively. No significant difference was observed in group demographics and comorbidities (p > 0.05). Fracture characteristics were similar between groups apart from tibiofibular syndesmosis injury, 62.5% (20/32) in COVID-19 vs 42.0% (42/100) in 2018 (p = 0.03). Time-to-surgery was not significantly different between the two groups . Mean visual analog scale scores, ankle strength, and ankle range of motion in plantarflexion were not significantly different between the two groups at three and six months postoperatively (p > 0.05). Dorsiflexion was significantly higher in the COVID-19 group at three months (p = 0.03), but not six months (p = 0.94) postoperatively. No significant difference in postoperative complication was seen between groups, 25.0% (8/32) COVID-19 group compared to 15.0% (15/100) 2018 group (p = 0.11).ConclusionsPatients who underwent surgery during the early months of the COVID-19 pandemic did not experience prolonged time-to-surgery and had similar outcomes compared to patients treated prior to the pandemic. Trimalleolar ankle fractures are fractures involving all three malleoli of the ankle. It is a relatively uncommon but severe type of ankle fracture, requiring prompt surgery in most cases . DelayedThe World Health Organization’s 2020 declaration of the COVID-19 pandemic resulted in drastic worldwide health system reorganization measures to stem the spread of the disease and conserve limited healthcare resources . As partTherefore, the aim of this study was to determine if the COVID-19 pandemic impacted surgical treatment of trimalleolar ankle fractures at a large, academic health system by evaluating (1) time-to-surgery, (2) complication rates, and (3) functional outcomes in patients who underwent open reduction and internal fixation (ORIF). We hypothesized that patients who underwent ORIF of trimalleolar ankle fractures during the COVID-19 pandemic experienced prolonged time-to-surgery, higher complication rates, and poorer functional outcomes when compared to pre-pandemic patients. We undertook a retrospective cohort study to compare time-to-surgery and outcomes in patients who had undergone surgery prior to the COVID-19 pandemic and during the COVID-19 pandemic.This was a retrospective cohort study consisting of two cohorts created according to their date of surgery. The COVID-19 pandemic cohort consisted of patients who underwent surgery at the immediate start of the pandemic from April 1 to July 31 of 2020 (COVID-19 group). The pre-pandemic cohort consisted of patients who underwent surgery prior to the pandemic from January 1 to December 31 of 2018 (2018 group). Patients ≥18 years of age who underwent surgical management for trimalleolar ankle fracture at our institution during the study period were eligible for inclusion. Patients without follow-up at three to six months postoperatively were excluded from the study.Following expedited institutional review board approval by Henry Ford Health System Institutional Review Board , patients who underwent ORIF for trimalleolar ankle fracture were identified by querying a billing record database for Current Procedural Terminology code 27822. A manual retrospective chart review was conducted by research assistants to obtain information from the medical charts of patients who met inclusion criteria for this study. Age, sex, race, body mass index, zip code, smoking history, and a prior diagnosis of type 2 diabetes mellitus were recorded. Zip code was cross-referenced with a United States Census Bureau website to determine median household income . PreoperStatistical analysis was performed using SAS 9.4  with significance set at p-value < 0.05. Sex, race, marital status, and complication rate were reported as frequency and percentages and analyzed using chi-square test. Age, median income, and time-to-surgery were reported as mean ± standard deviation and analyzed using two-sample T-test. Ankle strength, ankle ROM, and VAS score at three and six months postoperatively were analyzed using the Wilcoxon rank-sum test.Thirty-two patients  underwent ORIF for trimalleolar ankle fracture between April and July 2020 and met inclusion criteria for this study, compared to 100 patients  in 2018. The groups were not significantly different with regards to age, sex, race, income, marital status, smoking status, or incidence of diabetes (p > 0.05). Demographic information is presented in Table The most common mechanism of injury in both groups was fall, 84.3% 27/32) in the COVID-19 group vs 84.0% in the 2018 group (84/100). Mechanism of injury did not significantly differ between the two groups (p = 0.74). Comparison of ankle fracture characteristics revealed similar frequencies of open fractures  and comminution of the posterior malleolus  between the groups, but a higher frequency of tibiofibular syndesmosis injury in the COVID-19 group . There was no significant difference between the COVID-19 group and the 2018 group when comparing the rates of additional fractures other than ankle fracture, 21.9% (7/32) and 18.0% (18/100), respectively (p = 0.57). Similarly, there was no significant difference in external fixation prior to definitive ORIF between the COVID-19 group and the 2018 group, 15.6% (5/32) compared to 12.0% (12/100), respectively (p = 0.53). Injury and fracture characteristics are presented in Table 7/32 in tTime-to-surgery was similar between groups, with an average of 8.84 ± 6.78 days for the COVID-19 group, compared to 8.61 ± 6.02 days in the 2018 group (p = 0.85). There was a 10% difference in complication rate between the two groups, with 25.0% 8/32) of patients in the COVID-19 group experiencing one or more complications related to their surgery up to one year postoperatively, compared to 15.0% (15/100) in the 2018 group. However, the difference in complication rate between the two groups was not statistically significant (p = 0.19). Postoperative complications and their frequencies are detailed in Table 2 of patiFunctional outcomes examined by this study were postoperative pain levels, ankle strength, and ankle ROM at three and six months postoperatively. Mean VAS pain score was not significantly different between the groups at three months  and six months  postoperatively. Strength of ankle dorsiflexion, plantarflexion, eversion, and inversion were not significantly different between the groups at three months  and six months  postoperatively. ROM in ankle dorsiflexion was significantly higher in the COVID-19 group at three months , but not six months postoperatively (p = 0.94). ROM in plantarflexion was not significantly different between the groups at three months (p = 0.96) and six months (p = 0.84) postoperatively.Considering the well-documented effect of seasonality on fracture frequencies and characteristics ,14, thisThe purpose of this study was to determine the impact of the COVID-19 pandemic on surgical timing and outcomes of ORIF for trimalleolar fractures at a large, academic health system. The study compared a cohort treated during the early months of the COVID-19 pandemic (COVID-19 group) to an equitable pre-pandemic cohort (2018 group). The number of patients in the COVID-19 group compared to the 2018 group was reduced possibly as a result of behavior or activity changes during the pandemic ,16. We fDemographically, we did not find any major differences between the 2018 and COVID-19 groups. Patients were similar regarding age, sex, race, income, marital status, smoking status, and incidence of diabetes. Additionally, there was no significant difference in mechanism of injury or the majority of ankle fracture characteristics between groups. However, the COVID-19 cohort had a higher frequency of tibiofibular syndesmosis injury in comparison to the 2018 cohort.An additional key finding from our study was that surgery for trimalleolar ankle fracture was not significantly delayed during the early months of the COVID-19 pandemic. The COVID-19 and 2018 groups underwent surgery 8.84 ± 6.78 and 8.61 ± 6.02 days from their date of injury, respectively (p = 0.85). Currently, there is no consensus and conflicting evidence in the scientific literature regarding optimal time to surgical fixation -19. A reAdditionally, to our knowledge, no study has previously examined time-to-surgery for trimalleolar ankle fractures within United States hospital systems during the pandemic. A study by Wong and Cheung analyzed 122 outpatient clinics in Hong Kong and demonstrated that patients did not endure longer wait times for emergency operations or consultations . A retroThis study has several limitations, the first being that this was a retrospective study, with all the inherent limitations related to the quality or process of record-keeping. Additionally, retrospective studies are susceptible to biases, such as selection bias . We workThis study concludes that the COVID-19 pandemic did not have a significant impact on trimalleolar fracture outcomes at our institution. Time-to-surgery was not significantly different in the pandemic group compared to the 2018 group. There were non-significant differences in VAS scores, ankle strength, and ankle ROM at three and six months postoperatively and complications up to one year postoperatively.


Methamphetamine (METH) is a psychostimulant that has harmful effects on all organs, the nervous system, cardiovascular system, and reproductive system. Since many METH consumers are young people of reproductive age, it poses a risk to the next generation of METH consumers. METH can pass through the placenta and is also secreted into breast milk. Melatonin (MLT) is the primary hormone of the pineal gland that regulates the circadian cycle, and it is also an antioxidant that can mitigate the effects of toxic substances. This study aims to investigate the protective effect of melatonin against the detrimental effects that METH has on the reproductive system of male newborns, whose mothers consumed METH during pregnancy and lactation. In the current study, 30 female adult balb/c mice were divided into three groups: control group, vehicle group that received normal saline, and the experimental group that received 5 mg/kg METH intraperitoneally during gestation and lactation. After lactation, the male offspring of each group were randomly divided into two subgroups, one of which received 10 mg/kg melatonin intragastrically for 21 days (corresponding to the lactation period of the mice) (METH-MLT) and the other did not (METH -D.W). After treatment, the mice were sacrificed and testicular tissue and epididymis were obtained for the following tests.The diameter of seminiferous tubules, SOD activity, total Thiol groups concentration, catalase activity, sperm count, and PCNA and CCND gene expression were significantly increased in the METH-MLT group compared with the METH-DW. Apoptotic cells and MDA level ameliorated in the METH-MLT group compared with METH-D.W, and testicular weight had no notable change.The current study represents that consumption of METH during pregnancy and lactation can have adverse effects on the histological and biochemical factors of testis and sperm parameters of male newborns, which can be mitigated by taking melatonin after the end of the breastfeeding period. Methamphetamine (METH) is a central nervous system stimulant that distributes more rapidly in the CNS than amphetamine, making it more pleasurable to drug users . The priMelatonin (MLT) is a hormone that is mainly produced by the pineal gland in the brain. It is a regulator of the circadian cycle which regulates the sleep-wake rhythm . It alsoAs there is insufficient evidence on the effects of METH usage during pregnancy and lactation on the reproductive system of male newborns and the curative effect of MLT after METH usage, in the current study, we will investigate the efficacy of consumption of MLT for mitigating the negative effects of METH abuse during pregnancy and lactation on the reproductive system of male newborns.°C, relative humidity of 55±5%, and a 12-hour light-dark cycle. The animals had free access to standard pellet feed and water.In this study, 30 female and 15 male adult BALB/c mice were provided by the animal care center of Mashhad University of Medical Sciences. The animals were kept in an air-conditioned animal care center at 23±2 Every two female mice were housed with one male mouse for mating at 5:00 PM. The following day, the female mice were checked, and the presence of a vaginal plug was determined to signify pregnancy day zero (0). Twenty-four pregnant female mice were randomly divided into three groups, including 1) the control group (Ctrl) without any intervention, 2) the saline  group that received saline, and 3) the METH group that received METH (5 mg/kg). METH was dissolved in normal saline and injected daily by intraperitoneal (IP) injection during pregnancy and lactation . After lAfter the treatment period, the animals were sacrificed to remove their testes and epididymis, left testis was fixed in 10% normaline  for histological study, and the right testis for testis weight, oxidative stress evaluation, and real-time PCR assessment.Testes weightThe right testes were weighed by a 0.1 mg precise digital scale.Biochemical measurementsFor biochemical measurements, testis tissues were homogenized. For this purpose, 1 ml of cold phosphate-buffered saline (PBS) was added to 100 mg of testes tissue, and the tissue was then homogenized by a mechanical homogenizer.Malondialdehyde (MDA)MDA level is an index of lipid peroxidation. A solution containing 375 mg of TBA, 15 mg of TCA, and 2 ml of HCl was prepared, and the total volume was made up to 100 ml. One milliliter of homogenized testis tissue was added to 2 ml of complex solution, and put in a boiling water bath for 45 min. Then it was placed at room temperature and after cooling it was centrifuged at 1000 rpm for 10 min, the supernatant was removed, and its absorbance was read at 535 nm, 19. The5C(M)=Absorbance ⁄1.65×10Superoxide dismutase (SOD) SOD activity was measured according to the procedure described by Madesh and Balasubramanian method. A colorimetric assay involving the production of superoxide through autoxidation of pyrogallol and inhibiting the reduction of MTT  2, 5-diphenyltetrazolium bromide) to formazan was performed. An appropriate amount of homogenized tissue, MTT, and pyrogallol was added to the plate and incubated in a dark place at room temperature. DMSO was added and shaken gently to inhibit the reaction. Finally, the absorbance was read at 570 nm using a microplate reader . One unit of SOD activity was defined as the amount of enzyme that caused 50% inhibition of the MTT reduction rate .Measurement of total thiol groups (SH)-1cm-1. A mixture of 1 ml of Tris-EDTA buffer (pH=8.6) and 50 μl of tissue homogenate was prepared and its absorbance was read at 412 nm against a blank (A1). Then, 20 μl of DTNB reagent was added to this mixture, and after 10 min of incubation at room temperature, absorbance was read again (A2). The absorbance of the DTNB reagent was read as blank (B). The total thiol concentration (mM) was calculated according to the following formula ×1.07/0.05×13.6Total thiol concentration (mM)= assay2O2. Using the slope of the H2O2 absorption curve, CAT enzyme activity was calculated and normalized to protein concentration  according to standard histological methods. After fixation in normalin for two days at room temperature, specimens were dehydrated with ascending graded alcohol, cleared with xylene, and then embedded in paraffin. The paraffin blocks were cut into 5 µm thickness by a rotatory Leitz microtome, and finally, the tissue sections were stained with H&E.To measure the seminiferous tubule diameter, five photographs were randomly taken from each slide using ×40 objective lens of a light microscope  equipped with a high-resolution camera (DP10). Each image was analyzed using the image processing software analySIS LS Starter. Tubule diameter was measured in the cross sections of seminiferous tubules .TUNEL methodThe terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method was used to detect apoptotic cells using the TUNEL kit . 2O2, and after washing, 15 μl of protein kinase was added for 20 min, after washing again, the enzyme and enzyme label were added and incubated for 24 hr in the refrigerator. After washing again, 15 μl of POD (horseradish peroxidase) was add, one hour of incubation was done, then 15 μl of DAB was added to the sections and then stained with hematoxylin. In this method, apoptotic cells are detected by dark brown nuclei. Tissue sections were evaluated and photographed using ×40 objective lens of a light microscope . The number of TUNEL-positive cells was counted and calculated according to the following formula  at 40x_ magnification, and the average amount of sperm counted was reported. The number of sperm per milliliter was calculated using the following formula:Sperm morphology Papanicolaou staining was performed to study sperm morphology. For this purpose, 50 μl of sperm solution was smeared on a glass slide and fixed with methanol (70%), then the slide was stained. On each slide, 200 sperms were counted under a light microscope (40x magnification). The sperms without tails, with curled or bent tails, or with two or abnormal heads were recognized as abnormal sperms.DNA Fragmentation Index (DFI)6 /ml, 50 ul of sperm solution was mixed with melted agarose, and 30 µl of the cell suspension was placed on a coated slide glass (included in the kit) and covered with a coverslip and incubated at 4 °C. After 5 min, the coverslip was carefully removed, and the denaturing solution was added and incubated for 7 min at room temperature. Then the slides were immersed in a lysis solution. After 15 min, the glass slides were washed with distilled water. The glass slides were dehydrated with alcohol and then stained using the kit protocol. Sperm DNA fragmentation was assessed using the SDFA kit  according to the manufacturer’s instructions. The sperm count reached 5-10×10After covering the slide with a coverslip, the spermatozoa were observed under a light microscope at 40 magnification. DNA fragmentation was classified based on spermatozoa halo size. Spermatozoa with a large or medium halo were scored as having no fragmentation , and spermatozoa with a small or no halo were scored as indicating fragmentation (DNA-fragmented spermatozoa). A total of 200 spermatozoa were examined and scored in each sample. The fragmentation rate was calculated as DFI (%)=×100 .Quantitative real-time PCR-ΔΔCt method. Standard curves for the target  and reference (GAPDH) genes were generated  according to the manufacturer’s protocol. Purification and concentration of the extracted RNAs were measured using NanoDrop 2000C . One microliter of the extracted RNA was used for cDNA synthesis based on the manufacturer’s instructions  . Quantitenerated .Statistical analysispost hoc. Data were expressed as mean±SD, and P-values less than 0.05 were considered statistically significant.Statistical analysis was performed using Prism v.8.4.3 software (GraphPad Software). Data were analyzed using one-way ANOVA and Tukey’s Testes weightThere was no significant difference in testis weight between the studied groups .Seminiferous tubule diameterP≤0.001). The METH-MLT group had no significant difference compared with the Ctrl-D.W and Sal-D.W groups , and also compared with METH-MLT (P≤0.05), and there were no significant differences between other groups. MDA level in the METH-D.W group was significantly increased compared with Ctrl-D.W and Ctrl-MLT and Sal. D.W, and Sal-MLT groups (SODP≤0.05), and also in comparison with Ctrl-D.W and Ctrl-MLT, and Sal. D.W and Sal-MLT groups (P≤0.001), but no significant differences were observed between other groups (The SOD activity in the METH-D.W group decreased compared with METH-MLT (r groups .Thiol groups P≤0.001) and METH-MLT (P≤0.01). There was no significant difference between the other groups , and Sal. D.W and Sal-MLT groups (P≤0.01). The CAT activity in the METH-MLT group was higher than in the METH-D.W group (P≤0.05) .The CAT activity decreased in the METH-D.W group compared with Ctrl-D.W and Ctrl-MLT groups (TUNEL assay P≤0.001), and in METH-MLT group was lower than in METH-D.W group (P≤0.01), ,  and 5. Sperm parametersSperm countP≤0.001). Sperm count was significantly increased in the METH-MLT group compared with the METH-D.W group P≤0.001). This parameter was decreased in the METH-MLT group compared with the METH-D.W group (P≤0.01). The METH-MLT group had no significant difference in comparison with Ctrl-D.W and Sal-D.W, but it was significantly decreased compared with Ctrl-MLT and Sal-MLT groups (P≤0.001), , .Real-Time PCRPCNAP≤0.001) and increased in METH-MLT compared with METH-D.W (P≤0.001). There was no significant difference in the expression of this gene in the METH-MLT group compared with the Ctrl-D.W group , and the expression of this gene was significantly increased in the METH-MLT group compared with the METH-D.W group (P≤0.001), but there was no significant difference in comparison with Ctrl-D.W group .The authors would like to appreciate the Not applicable.Consent for publication is given by all authors. The authors declare that they have no competing interests.